Effective silencing of ENaC by siRNA delivered with epithelial-targeted nanocomplexes in human cystic fibrosis cells and in mouse lung

Abstract

Introduction: Loss of the cystic fibrosis transmembrane conductance regulator in cystic fibrosis (CF) leads to hyperabsorption of sodium and fluid from the airway due to upregulation of the epithelial sodium channel (ENaC). Thickened mucus and depleted airway surface liquid (ASL) then lead to impaired mucociliary clearance. ENaC regulation is thus a promising target for CF therapy. Our aim was to develop siRNA nanocomplexes that mediate effective silencing of airway epithelial ENaC in vitro and in vivo with functional correction of epithelial ion and fluid transport.

Methods: We investigated translocation of nanocomplexes through mucus and their transfection efficiency in primary CF epithelial cells grown at air-liquid interface (ALI).Short interfering RNA (SiRNA)-mediated silencing was examined by quantitative RT-PCR and western analysis of ENaC. Transepithelial potential (V_t), short circuit current (I_sc), ASL depth and ciliary beat frequency (CBF) were measured for functional analysis. Inflammation was analysed by histological analysis of normal mouse lung tissue sections.

Results: Nanocomplexes translocated more rapidly than siRNA alone through mucus. Transfections of primary CF epithelial cells with nanocomplexes targeting αENaC siRNA, reduced αENaC and βENaC mRNA by 30%. Transfections reduced V_t, the amiloride-sensitive I_sc and mucus protein concentration while increasing ASL depth and CBF to normal levels. A single dose of siRNA in mouse lung silenced ENaC by approximately 30%, which persisted for at least 7 days. Three doses of siRNA increased silencing to approximately 50%.

Conclusion: Nanoparticle-mediated delivery of ENaCsiRNA to ALI cultures corrected aspects of the mucociliary defect in human CF cells and offers effective delivery and silencing in vivo.

Keywords: airway epithelium; cystic fibrosis; lung physiology; nebuliser therapy.

Figure 1

Nanoparticles translocate vertically through the mucus barrier. Cy3-siRNA alone, RTNs containing Cy3-siRNA or fluorescent polystyrene (PS) nanoparticles were investigated for their translocation potential through (A) pig gastric mucus, (B) normal human airway mucus and (C) CF human airway mucus. The siRNA that is labelled with Cy3 is targeting glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The experiments were repeated on three occasions, and each point is the mean±SEM of triplicate measurements. CF, cystic fibrosis; RTNs, receptor-targeted nanocomplexes; siRNA, short interfering RNA.

Figure 2

ENaC expression over time of primary cells growing at ALI and silencing of ENaC. (A) The fold-difference in endogenous αENaC gene expression relative to β-actin was quantified by qRT-PCR in CFBE cells from the start date of ALI cultures (mean of n=3 per time point). Points represent mean values±SEM. (B) RTN formulations containing either αENaC siRNA or control siRNA at 100 nM were used in transfections of CFBE cells grown at ALI for 6 weeks. Transfections were performed once (single) or sequentially every other day (triple), and the percentage of silencing was calculated 48 hours after the last transfection (n=3 per formulation). The middle horizontal lines represent the median values. Silencing is normalised to the mean control siRNA set at 100%. (C) CFBE cells grown at ALI were triple-transfected with RTN formulations containing either αENaC siRNA or control siRNA at 100 nM, and the percentage of silencing of α, β and γ ENaC subunits was then calculated 48 hours after transfection (n=3 per formulation). The middle horizontal lines represent the median values. Silencing is normalised to the mean control siRNA set at 100%. Non-parametric Mann-Whitney U tests were performed, and no statistical significant differences were achieved. ALI, air–liquid interface; CFBE, cystic fibrosis bronchial epithelial cells; ENaC, epithelial sodium channel; RTN, receptor-targeted nanocomplex; siRNA, short interfering RNA.

Figure 3

Single transfection of CFBE air–liquid interface (ALI) cell monolayers with nanocomplexes containing αENaC siRNA reduces the amiloride-sensitive short circuit current (I_sc). (A) A schematic highlighting experiments performed after single or triple transfections on CFBE cells grown at ALI. (B) Representative I_sc traces from CFBE monolayers in Ussing chambers of cells treated with 100 nM αENaC siRNA or control siRNA or untreated cells. (C) The change in I_sc (ΔI_sc) after application of 10 µM amiloride is shown for the αENaC siRNA-treated cells (Ussing chambers; n=6), the control siRNA-treated cells (n=8) and the untreated CFBE cells (n=5). Median values are presented as bars and IQR by upper and lower horizontal lines, with statistical significance determined by the non-parametric Mann-Whitney U test. Asterisks indicate comparisons of specific formulations with statistical significance (*p<0.05; **p<0.01). (D) Transepithelial potential (V_t) of αENaC siRNA-treated monolayers of CFBE cells cultured for 4 weeks on ALI. Wells were triple-transfected with 100 nM αENaC siRNA (n=5) or control siRNA (n=5) and then V_t measurements performed 3 days after the last transfection using scanning ion-conductance microscopy (SICM). Further samples were treated with 10 µM VX-809 1 day prior to V_t measurement and treated with 10 µM VX-770 during measurement (for approximately 20 min; n=4). Median values are presented as bars and IQR by upper and lower horizontal lines. Non-parametric Mann-Whitney U tests were performed, and no statistical significant differences were achieved. ASL, airway surface liquid; CBF, ciliary beat frequency; CFBE, cystic fibrosis bronchial epithelial cells; ENaC, epithelial sodium channel; siRNA, short interfering RNA.

Figure 4

Effect of triple transfections of CFBE ALI cells with nanocomplexes containing αENaC siRNA. The CFBE cells were cultured in snapwells for 4 weeks then treated with 100 nM αENaC siRNA or control siRNA at 48 hours intervals and the measurements performed 3 days after the third dose. (A) ASL depth measurements of CFBE monolayers as determined by confocal microscopy. XZ representative images of fluorescently labelled ASL (red) and cells (green). Top: untreated cells, middle: cells transfected three times with 100 nM of control siRNA and bottom: cells transfected three times with 100 nM of αENaC siRNA. A white bar has been included to denote the ASL measurement. (B) ASL depth measurement of each treatment group (n=4). Bars represent mean±SEM, while asterisks indicate statistical significance of comparisons between the αENaC siRNA-treated cells versus the controls (***p<0.001) determined by an ANOVA test followed by Bonferroni’s post hoc test. (C) Effect of αENaC siRNA, control siRNA and VX-770 and VX-809 on ciliary beat frequency (CBF) of CFBE cells grown at ALI. For each experimental condition (n=10), readings of CBF were calculated from 10 ciliated areas in the snapwell, and the data represent the mean±SEM. Asterisks indicate comparisons of specific formulations with statistical significance (*p<0.05; **p<0.01; ***p<0.001) determined by an ANOVA test followed by Bonferroni’s post hoc test. (D) Transepithelial fluid transport through human CFBE cells. Net fluid absorption rate was measured 4 days after cell treatment with a control siRNA or with αENaC siRNA. Bars represent the median value with IQR shown by the horizontal lines (n=4), while asterisks indicate statistical significance (*p<0.05) determined by non-parametric Mann-Whitney U tests. ALI, air–liquid interface; ASL, airway surface liquid; CFBE, cystic fibrosis bronchial epithelial cells; ENaC, epithelial sodium channel; siRNA, short interfering RNA.

Figure 5

Protein concentration in mucus collected at different time points from transfected or untreated CFBE cells grown at ALI and in vitro silencing after three transfections. Mucus collections were done (A) 2 days after the first transfection (αENaC siRNA treated, n=6; control siRNA treated, n=3; untreated controls, n=3), (B) 2 days after the second transfection (αENaC siRNA treated, n=6; control siRNA treated, n=3; VX-770 and VX-809 treated, n=3; untreated controls, n=3) and (C) 7 days after the third transfection (αENaC siRNA treated, n=6; control siRNA treated, n=3; VX-770 and VX-809 treated, n=3; untreated controls, n=3). The concentration levels were normalised to those of the untreated cells. (D) Formulations containing either 100 nM αENaC siRNA (n=6) or control siRNA (n=3) were used in three sequential transfections of CFBE-BMI-1 cells grown at ALI for 4 weeks, and the percentage of silencing was calculated 8 days after the third transfection. Silencing was normalised to the mean control siRNA set at 100%. The middle horizontal lines represent the median values. Asterisks indicate comparisons of specific formulations with statistical significance (*p<0.05) determined by non-parametric Mann-Whitney U tests. ALI, air–liquid interface; CFBE, cystic fibrosis bronchial epithelial cells; ENaC, epithelial sodium channel; siRNA, short interfering RNA.

Figure 6

In vivo lung administration of siRNA-containing nanocomplexes. (A) Uptake of Dy677-siRNA formulations following oropharyngeal administration. Twenty-four hours later, the mice were culled (n=3 per group), and organs (heart, lung, liver, kidneys, spleen and intestines) were extracted and imaged for fluorescence. (B–D) The remaining αENaC mRNA was quantified by qRT-PCR in C57BL6 female mice after instillation of cationic nanocomplexes containing 16 µg αENaC siRNA (n=7) or control siRNA (n=6) at (B) 48 hours and (C) 7 days. (D) The amount of remaining αENaC mRNA detected by qRT-PCR in C57BL6 female mice at 72 hours after the last of three instillations of cationic nanocomplexes containing 16 µg αENaC siRNA (n=8) or control siRNA (n=7). Silencing was normalised to the mean control siRNA set at 100%. Medians and IQRs are presented by horizontal lines. Asterisks indicate comparisons of specific formulations with statistical significance (*p<0.05; **p<0.01; ***p<0.001) determined by non-parametric Mann-Whitney U tests. ENaC, epithelial sodium channel; siRNA, short interfering RNA.

Figure 7

In vivo delivery of siRNA-containing nanocomplexes. Representative images of H&E stained murine lung sections following single (A) and triple (B) instillations of cationic αENaC siRNA nanocomplexes (n=3 for both), or a triple instillation (n=3) of cationic control siRNA nanocomplexes (C). All triple-instilled treated mice received three doses of nanocomplexes containing 16 µg siRNA on alternate days, and the lungs were harvested 48 hours after the third instillation. The mice that had one instillation of nanocomplexes containing 16 µg siRNA, also had their lungs harvested 48 hours following instillation. Scale bars=100 µm. siRNA, short interfering RNA.